| Gastric cancer is one of the most prevalent malignancies, and chemotherapy remains the conventional method in treating it. However, long-term exposure to chemotherapeutic drugs often confers tumor cells resistance to a number of diverse natural product drugs that do not share a common structure or target, which is termed as multidrug resistance (MDR). Resistance to chemotherapeutic drugs is the toughest problem in cancer management. Not surprisingly, to improve the prognosis and treatment of cancer patients, it's necessary to understand the molecular mechanisms underlying the tumor MDR.In our previous work, we had established four resistant sublines from human gastric cancer cell line SGC7901. Studies revealed that MDRl/Pgp-mediated classical MDR only in the vincristine-induced drug-resistant cells. Chromosome G region analysis indicated that the structural alterations in chroumosome 6. By subtractive hybridization analysis of the SGC7901 and its drug-resistant cell line, we found that theZNRDl (locus chromosome 6p21.3) was only overexpressed in the S GC7901/VCRcell line as compared with its parental cell line. In this study, we confirmed the differential expression of ZNRD1 in SGC7901/VCR and its sensitive counterpart, and also explored the function and possible mechanisms of ZNRD1 related drug resistance, all these laid a solid basis for reverse of MDR in gastric cancer.Aims: To clone the encoding gene of ZNRD1, identify the differential expression of ZNRD1 in SGC7901/VCR and investigate the function and possible mechanisms of ZNRD1 related drug resistance.Methods: (1) Northern blot and semiquantitative RT-PCR assay were used to identify the differential expression of ZNRD1 RNA in SGC7901/VCR and its sensitive counterpart; (2) The encoding gene of ZNRD1 from SGC7901/VCR cell was amplified using RT-PCR; (3) The cDNA fragments were subcloned into prokaryotic expression vector and expressed in E coli; (4) Prokaryotic expressed protein products of ZNRD1 cDNAs were purified by Ni-NTA and used as immunogens to prepare rabbit antisera; (5) The antisera were characterized by Western blot; (6) Immunochemical method was used to detect the expression of protein in SGC7901/VCR and its sensitive counterpart; (7) The antisense RNA expression vector according to ZNRD1 gene was constructed using DNA recombinant technique and transfected into SGC7901/VCR cells by lipofectamine. ZNRD1 expression was confirmed in transfected cells by Dot hybridization; (8) Cell cycle analysis was performed using flow cytometry; (9) Growth curve and drug sensitivity assay were performed using MTT assay. IC50 values and resistance index (RI) of gastric cancer cells to anticancer drugs were calculated; (10) Eukaryotic vector was constructed and transfected into SGC7901 cells by microinjection. ZNRD1 expression wasconfirmed in transfected cells by Dot hybridization; (11) Drug sensitivity assay were performed using MTT assay; (12) The intracellular adriamycin accumulation of SGC7901/VCR cells and transfectants was determined using flow cytometry; (13) The expression of MDR1 , MHRP, GST- , MGr1 in cells were analyzed using Western blot.Results: (1) As Northern blot and semiquantitative RT-PCR discovered, the RNA expression levels of ZNRD1 in SGC7901/VCR cells is much higher than that in SGC7901 cells; (2) The fusion protein was highly expressed in E.Coli and that the comprises 32% of total bacterial protein. The polyclonal antibody was successfully prepared with the purified recombinant ZNRD1 and recognized a special protein band of ZNRD1; (3) Immunochemical results showed that ZNRD1 protein was expressed mostly in the nucleus, and the expression level was higher in SGC7901/VCR cells than that in SGC7901 cells; (4) As suggested by Dot hybridization, the expression level of ZNRD1 in SGC7901/VCR cells decreased significantly after transfected with ZNRD1 antisense RNA expression vector; (5) As showed by flow cytometry, ZNRD1 down-regulated gastric cancer cells SGC7901/VCR-an ZNRD1 exhibited slower proliferation than SGC7901...
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